Optimising reproductive induction in yellowbelly flounder (Rhombosolea leporina): Evaluating GnRHa dosage and non-invasive gonadal assessment

Abstract

This study investigates two complementary tools to address reproductive dysfunction in wild-caught yellowbelly flounder (Rhombosolea leporina), a key bottleneck in the species’ aquaculture development. First, the effects of varying doses of gonadotropin-releasing hormone analogue (GnRHa) on ovarian development and expression of gonadal cyp19a1a, a gene encoding aromatase, critical for 17β-estradiol synthesis were quantified at short-term timepoints. A single injection of 50 or 100 µg/kg induced transient upregulation of cyp19a1a by Day 1 post-treatment (log₂ fold-changes of +2.38 and +2.65, respectively), while the 25 µg/kg and control groups showed downregulation (−0.53 and −0.07) relative to Day 0 baselines. However, expression declined significantly in all groups by Day 5, including a drop in the 100 µg/kg group to −0.68 (p = 0.03), indicating a brief stimulatory window likely insufficient to sustain steroidogenic activity or promote meaningful oocyte development. These findings support the need for sustained-release hormone systems or improved broodstock staging to enhance treatment efficacy.

Second, a novel, non-invasive Visual Gonadosomatic Index (VGSI) was evaluated to minimise handling stress while classifying reproductive condition and suitability for hormone treatment. Two metrics were developed using backlit imaging of the ventral gonads: gonad area relative to body area (Size GSI) and gonad length relative to body length (Length GSI). While neither metric correlated strongly with oocyte diameter or histological stage, Length GSI showed a modest but significant association with oocyte development (R² = 0.1937, p = 0.009), and both indices were positively associated with cyp19a1a expression (Size GSI: R² = 0.5045, p = 0.021; Length GSI: R² = 0.3408, p = 0.076). These results suggest VGSI may provide a coarse proxy for reproductive development, though further refinement and validation are required.

Together, these findings represent the first report of ovarian cyp19a1a expression in R. leporina and lay the groundwork for integrated hormonal and imaging-based strategies to improve reproductive control in this culturally and economically significant flatfish.